Most physiological and behavioral processes exhibit daily oscillations under the control of an internal circadian timing system, and disruptions of this system cause metabolic and cognitive deficits in diverse organisms, including humans. The circadian system is composed of dedicated clock neurons, which contain cell autonomous molecular clocks, input pathways, which synchronize these clocks to external signals such as light, and output pathways, which allow circadian signals to coordinately influence physiology and behavior. Though much is known about the molecular components of the circadian clock, as well as the identity of the core clock neurons, little is known about the downstream neuronal populations that comprise the circadian output pathway. The overall aim of this project is to determine, on a circuit level, how the information derived from molecular clocks in individual neurons is coordinated across neuronal populations within the clock network, and how this information is translated into coherent behavioral outputs. Specific Aim 1 incorporates novel genetic and anatomical techniques to map the synaptic partners of clock neurons of the fruit fly, Drosophila melanogaster, thereby identifying the anatomical substrate through which circadian signals are translated into rhythmic behavioral outputs. Specific Aim 2 takes advantage of the power of Drosophila genetics to identify neuronal populations that are part of the circadian output circuit. Through the use genetic tools that allow for combined spatial and temporal control over neuronal excitability, neuronal populations will be identified in which constitutive activation degrades rest:activity rhythms. This unbiased approach will identify not only neurons directly contacted by clock cells, but also populations further downstream of circadian inputs. Finally, Specific Aim 3 seeks to determine the locus of action of the known circadian output gene, Neurofibromatosis 1, as well as downstream components of its signaling cascade. This will link specific molecular components of the output pathway with the circadian output circuit, and will also serve as independent confirmation that the neuronal populations identified in Specific Aim 2 comprise part of the circadian output circuit. These experiments will lay the groundwork for a combined cellular and molecular understanding of the mechanisms through which the circadian system influences physiological and behavioral processes, and will therefore provide a basis for understanding the health problems associated with circadian dysfunction.